Particle and pharmaceutical composition comprising an insoluble camptothecin compound with double core-shell structure and method for manufacturing the same

ABSTRACT

The present invention relates to a drug delivery system having a double core-shell structure and, specifically, to a double nano-drug delivery system having an inner core-shell containing a poorly soluble camptothecin compound and a water-soluble camptothecin compound inside and an amphiphilic polymer shell, and to a manufacturing method therefor. The double core-shell structured particles manufactured by the present invention form very stable particles and show a mono-distribution of particles before and after freeze-drying. The particles of the present invention show excellent results compared with existing monolayer micelles in animal efficacy tests and pharmacokinetic tests, and do not use a surfactant causing hypersensitivity, and thus the use of the particles of the present invention can provide a pharmaceutical composition or a drug delivery system platform, which are safe for the human body.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of Korean PatentApplication No. 10-2017-0079354 filed in the Korean IntellectualProperty Office on 22 Jun. 2017, the disclosure of which is incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to a drug delivery system having a doublecore-shell structure and, specifically, to a double nano-drug deliverysystem having an inner core-shell containing a poorly solublecamptothecin compound and a water-soluble camptothecin compound insideand an amphiphilic polymer shell, and to a manufacturing methodtherefor.

BACKGROUND

A solubilizing step is essentially needed to develop a poorly solubledrug to be used for injection, and in order to overcome such a problem,emulsions containing surfactants, micro-emulsions, liposomes, micelles,pegylation, or making produgs, have been employed. 7-Ethyl 10-hydroxycamptothecin (SN-38), which is one of the drugs having strongestactivity as camptothecin-based anticancer drugs, is supplied asirinotecan in a prodrug form (trade name: CAMTOSAR®, manufactured byNovartis), which is mainly hydrolyzed by carboxylesterase II (CESII) invivo to be converted into SN-38 in an active form. However, the rate ofconversion is as very low as 2-8% and the deviation thereof is also verylarge, and thus such a drug has difficulty in proper administration tocancer patients in need of precise dosage adjustment, with the resultthat the effects and side effects thereof are difficult to predict.

Another method for solubilization of a poorly soluble drug is tomicellize a drug to have a nano-sized particle diameter. In order toallow a nanomicelle injection to secure sterility and stability, ananomicelle aqueous solution is sterile-filtered through a 0.22-μmfilter, and then powdered into solid through freeze-drying, during themanufacturing process. In this process, when a freeze-dried product isagain dissolved in a solvent for injection (saline or the like), a largeamount of macroparticles (200 nm or more to several tens of μm) may begenerated due to the agglomeration of micelles themselves. Inparticular, particles larger than 5 μm may cause severe side effectswhen injected into the human body, and thus are strictly managed throughinsoluble particulate matter tests in US Pharmacopoeia, EuropeanPharmacopoeia, and Korean Pharmacopoeia. Therefore, a solubilizingmethod whereby the particle sizes are little changed before and afterfreeze-drying in spite of micellization, particles show amono-distribution, and especially, there are no particles of several μmor more.

Meanwhile, it is essential to dissolve an active ingredient in anappropriate solvent in order to prepare an injection, but camptothecinor SN-38, which is a hydrophobic cam ptothecin-based compound, isfavorably dissolved in neither water nor most volatile polar organicsolvents (methanol, ethanol, acetonitrile, ethyl acetate, etc.) used inpharmaceutical preparation procedures. Solvents capable of dissolvingsuch the hydrophobic camptothecin-based compounds therein are limited tonon-volatile solvents, such as dimethyl sulfoxide (DMSO), dimethylformamide, toluene, and dioxane. However, a dialysis procedure is neededto remove these solvents, and these solvents are difficult to removecompletely in spite of dialysis, causing toxic problems when remaining.

Therefore, the present inventors searched and endeavored to developstable particle compositions whereby: camptothecin and SN-38, which areseverely poorly soluble anticancer active ingredients, can be directlyadministered in an active form but not a prodrug form; there are noproblems of residual solvents during the preparation process; there islittle change in particle size before and after freeze-drying; andmacro-agglomerated particles of several μm or more are not generated.

Throughout the entire specification, many patent documents arereferenced and their citations are represented. The disclosures of citedpapers and patent documents are entirely incorporated by reference intothe present specification, and the level of the technical field withinwhich the present invention falls and details of the present inventionare explained more clearly.

SUMMARY Technical Problem

An aspect of the present invention is to provide a particle including:i) a hydrophobic camptothecin-based compound; ii) a hydrophiliccamptothecin-based compound; and iii) an amphiphilic block copolymercomposed of a hydrophobic block and a hydrophilic block.

Another aspect of the present invention is to provide a pharmaceuticalcomposition for treating cancer containing the particle and apharmaceutically acceptable carrier.

Still another aspect of the present invention is to provide a method formanufacturing a particle, the method including:

(a) forming an inner core-shell containing a hydrophobiccamptothecin-based compound and a hydrophilic camptothecin-basedcompound; and (b) forming an outer core-shell containing an amphiphiliccopolymer.

Other purposes and advantages of the present disclosure will become moreobvious with the following detailed description of the invention,claims, and drawings.

Technical Solution

In accordance with an aspect of the present invention, there is providedinventions 1 to 35 below:

1. A particle comprising:

i) a hydrophobic camptothecin-based compound;

ii) a hydrophilic camptothecin-based compound; and

iii) an amphiphilic block copolymer composed of a hydrophobic block anda hydrophilic block.

2. The particle of claim 1, wherein the hydrophobic camptothecin-basedcompound is at least one selected from the group consisting of7-ethyl-10-hydroxycamptothecin (S N-38), cam ptothecin,10-hydroxycamptothecin, and a pharmaceutical acceptable salt thereof.

3. The particle of claim 1, wherein the hydrophilic camptothecin-basedcompound is at least one selected from irinotecan, topotecan, belotecan,exatecan, lurtotecan, sinotecan, rubitecan, 9-nitrocamptothecin, 9-aminocamptothecin, gimatecan, BNP-1530, DB-67, BN-80915, BN-80927, apharmaceutically acceptable salt thereof, a glucuronide metabolitethereof, and a glucuronide metabolite of the hydrophobiccamptothecin-based compound.

4. The particle of claim 1, wherein the amphiphilic block copolymer iscomposed of A-B or A-B-A blocks,

(a) wherein A is a hydrophilic polymer, which is monomethoxypolyethylene glycol, dimethoxy polyethylene glycol, polyethylene glycol,polypropylene glycol, monomethoxy polypropylene glycol, polyethyleneoxide, polyacrylic acid, or a polymer thereof; and

(b) wherein B is a hydrophobic polymer, which is polylactic acid,polylactide, polyglycolic acid, polyglycolide, a polylacticacid-co-glycolic acid copolymer, polymandelic acid, polycaprolactone,polydioxan-2-one, polyglutamic acid, polyaspartic acid, polyornithine,polyorthoester, a derivative thereof, or a copolymer of two or morecompounds selected therefrom.

5. The particle of claim 4, wherein the number average molecular weightof the hydrophilic polymer A is 500-10,000 Da.

6. The particle of claim 4, wherein the number average molecular weightof the hydrophobic polymer B is 500-10,000 Da.

7. The particle of claim 1, wherein the weight ratio of the hydrophobiccamptothecin-based compound and the hydrophilic camptothecin-basedcompound is 1:10 to 10:1.

8. The particle of claim 1, wherein the weight ratio of the sum of thehydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound and the amphiphilic block copolymer is 1:200to 10:1.

9. The particle of claim 1, wherein the number average particle size ofthe particle is 10-500 nm.

10. The particle of claim 1, wherein the particle has a doublecore-shell structure:

(a) an inner core-shell containing the hydrophilic camptothecin-basedcompound and the hydrophobic cam ptothecin-based compound; and

(b) an outer core-shell containing the amphiphilic block copolymer.

11. The particle of claim 1, wherein the particle has a bilayer micellestructure.

12. A pharmaceutical composition for treating cancer, comprising theparticle of any one of claims 1 to 11 and a pharmaceutically acceptablecarrier.

13. The composition of claim 12, wherein the cancer is selected from thegroup consisting of gastric cancer, ovarian cancer, uterine cancer,small cell lung cancer, non-small cell lung cancer, pancreatic cancer,breast cancer, esophageal cancer, oral cancer, rectal cancer, coloncancer, large intestine cancer, kidney cancer, prostate cancer,melanoma, liver cancer, gall bladder and other biliary tract cancer,thyroid cancer, bladder cancer, brain and central nervous system cancer,bone cancer, skin cancer, non-Hodgkin's and Hodgkin's lymphoma, andblood cancer.

14. The composition of claim 12, wherein the composition furthercomprises different types of anticancer drugs.

15. The composition of claim 12, further comprises sucrose, mannitol,sorbitol, glycerin, trehalose, and a polyethylene glycol excipient, anda cyclodextrin excipient.

16. A method for treating cancer, the method comprising administeringthe pharmaceutical composition of any one of claims 1 to 15 to asubject.

17. The method of claim 16, wherein the subject is human, mouse, rat,guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, orrhesus monkey.

18. A method for manufacturing a particle, the method comprising:

(a) forming an inner core-shell containing a hydrophobiccamptothecin-based compound and a hydrophilic camptothecin-basedcompound; and

(b) forming an outer core-shell containing an amphiphilic copolymer.

19. The method of claim 18, wherein step (a) comprises mixing thehydrophobic camptothecin-based compound and a hydrophiliccamptothecin-based compound in an organic solvent; and

wherein step (b) comprises mixing the inner core-shell and theamphiphilic block copolymer in an aqueous solvent.

20. The method of claim 18, wherein step (a) comprises mixing a basicaqueous solution, in which a hydrophobic camptothecin compound isdissolved, and an aqueous solution, in which a hydrophilic camptothecincompound is dissolved; and

wherein step (b) comprises mixing the inner core-shell and theamphiphilic block copolymer in an aqueous solvent.

21. The method of claim 20, wherein the aqueous solution in which thehydrophilic camptothecin compound is dissolved is a basic, neutral, oracidic aqueous solution.

22. The method of claim 20, wherein step (a) comprises:

(a1) mixing a basic aqueous solution, in which a hydrophobiccamptothecin-based compound is dissolved, and a basic, neutral, oracidic aqueous solution, in which a hydrophilic camptothecin-basedcompound is dissolved; and

(a2) lowering the pH of the mixed aqueous solution to 7 or lower.

23. The method of claim 18, wherein step (a) comprises mixing thehydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound in an organic solvent; and

wherein step (b) comprises mixing a mixture of the hydrophobiccamptothecin-based compound and the hydrophilic camptothecin-basedcompound with the amphiphilic block copolymer in an organic solvent.

24. The method of any one of claims 18 to 23, wherein the hydrophobiccamptothecin-based compound is at least one selected from the groupconsisting of 7-ethyl-10-hydroxycamptothecin (SN-38), cam ptothecin,10-hydroxycamptothecin, and a pharmaceutical acceptable salt thereof.

25. The method of any one of claims 18 to 24, wherein the hydrophiliccamptothecin-based compound is at least one selected from irinotecan,topotecan, belotecan, exatecan, lurtotecan, sinotecan, rubitecan,9-nitrocamptothecin, 9-aminocamptothecin, gimatecan, BNP-1530, DB-67,BN-80915, BN-80927, a pharmaceutically acceptable salt thereof, aglucuronide metabolite thereof, and a glucuronide metabolite of thehydrophobic camptothecin-based compound.

26. The method of any one of claims 18 to 24, wherein the amphiphilicblock copolymer is composed of A-B or A-B-A blocks,

(a) wherein A is a hydrophilic polymer, which is monomethoxypolyethylene glycol, dimethoxy polyethylene glycol, polyethylene glycol,polypropylene glycol, monomethoxy polypropylene glycol, polyethyleneoxide, polyacrylic acid, or a polymer thereof; and

(b) wherein B is a hydrophobic polymer, which is polylactic acid,polylactide, polyglycolic acid, polyglycolide, a polylacticacid-co-glycolic acid copolymer, polymandelic acid, polycaprolactone,polydioxan-2-one, polyglutamic acid, polyaspartic acid, polyornithine,polyorthoester, a derivative thereof, or a copolymer of two or morecompounds selected therefrom.

27. The method of claim 26, wherein the molecular weight of thehydrophilic polymer A is 500-10,000 Da.

28. The method of claim 26, wherein the molecular weight of thehydrophobic polymer B is 500-10,000 Da.

29. The method of any one of claims 18 to 24, wherein the weight ratioof the hydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound is 1:10 to 10:1.

30. The method of any one of claims 18 to 24, wherein the weight ratioof the sum of the hydrophobic camptothecin-based compound and thehydrophilic camptothecin-based compound and the amphiphilic blockcopolymer is 1:200 to 10:1.

31. The method of any one of claims 18 to 24, wherein the organicsolvent is a C1-05 alcohol (methanol, ethanol, propanol, butanol,n-butanol, iso-propanol, 1-pentanol, 2-butoxyethanol, isobutyl alcohol,etc.), an alkyl acetate, acetone, acetonitrile, chloroform, benzene,toluene, xylene, acetone, fluoroalkane, pentane, hexane,2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane,diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, diisopropylether, 2-chloropropane, 1-chloropropane, chlorobenzene, benzene, diethylether, diethyl sulfide, dichloromethane, 1,2-dichloroethane, aniline,diethyl amine, an ether, carbon tetrachloride, tetrahydrofuran (THF), ora mixed solvent thereof.

32. The method of claim 21 or 22, wherein the acidic aqueous solutionincludes at least one selected from pharmaceutically acceptableinorganic acids including hydrochloric acid, nitric acid, sulfuric acid,and phosphoric acid, or organic acids including citric acid, malic acid,lactic acid, acetic acid, and tartaric acid.

33. The method of claim 21 or 22, wherein the pH of the acidic aqueoussolution is 1.0 to 6.

34. The method of claim 20, wherein the basic solution includes at leastone selected from the group consisting of an inorganic alkali, an alkalisalt of an organic acid, and an alkyl amine, the inorganic alkaliincluding sodium hydroxide, potassium hydroxide, sodiumdihydrogenphosphate, potassium dihydrogenphosphate, magnesium hydroxide,sodium carbonate, and sodium hydrogencarbonate.

35. The method of claim 20, wherein the pH of the basic aqueous solutionis 8 to 13.

The present inventors endeavored to improve the solubility and stabilityof a hydrophobic camptothecin-based compound, of which the applicationis restricted due to severe poor solubility thereof, in spite of stronganticancer activity thereof, and as a result, the present inventorsconfirmed that the mixing of a hydrophobic camptothecin-based compoundand a hydrophilic camptothecin-based compound in an aqueous solvent ororganic solvent leads to a core-shell particle (micelle) having core andshell structures formed of hydrophobic- and hydrophilic-based compounds,respectively. Furthermore, it was confirmed that the addition of anamphiphilic block copolymer to the core-shell particle forms a doublecore-shell particle (bilayer micelle) in which the particle is enclosedin the amphiphilic block copolymer.

According to an embodiment of the present invention, the doublecore-shell particle containing both of a hydrophobic camptothecin-basedcompound and a hydrophilic camptothecin-based compound in the presentinvention has remarkably improved solubility compared with existingpoorly soluble camptothecin even without administration in the form of aprodrug. Therefore, the drug efficacy of active camptothecin (SN-38) canbe exerted regardless of the activity of carboxylesterase II (CESII) invivo. In addition, it was confirmed that the particles of the presentinvention caused no particle agglomeration or precipitation even thoughthe particles are again dissolved in an aqueous solvent afterfreeze-drying, and thus the particles had a dosage form with veryexcellent stability. Therefore, the present invention provides aparticle composition with a stable double core-shell structure, which isadvanced from an existing a monolayer nanomicelle to resolve a problemof low solubility of a poorly soluble drug.

In accordance with an aspect of the present invention, there is provideda particle comprising: i) a hydrophobic camptothecin-based compound; ii)a hydrophilic camptothecin-based compound; and iii) an amphiphilic blockcopolymer composed of a hydrophobic block and a hydrophilic block.

Herein, camptothecin is a topoisomerase inhibitor found in bark andstalk of the Camptotheca tree (Happy tree) and exhibits a very excellentanticancer effect in the preclinical stage, but cannot be used due tolow solubility thereof. Therefore, many researchers have developedcamptothecin analogues for improving the solubility of cam ptothecin.Currently, three types of cam ptothecin derivatives, irinotecan,topotecan, and belotecan, have been approved, and used in chemotherapyfor cancer.

As used herein, the term “hydrophobicity” refers to the exclusion fromwater molecules and agglomeration, as a tendency shown in non-polarmaterials. When a hydrophobic material is present in a hydrophilicliquid, the hydrophobic material agglomerates while increasinghydrophobic bonds as if the hydrophobic material is afraid of water.

As used herein, the term “hydrophilicity” refers to the property ofbeing dissolved in water with strong affinity with water, as a tendencyshown in polar materials. For example, a hydrophilic polymer compound,or a micelle colloid surface of a surfactant has strong hydrophilicity.

According to an embodiment, the hydrophobic camptothecin-based compoundis selected from the group consisting of 7-ethyl-10-hydroxycamptothecin(SN-38), camptothecin, 10-hydroxycamptothecin, and a pharmaceuticalacceptable salt thereof, but is not limited thereto.

According to another embodiment of the present invention, thehydrophilic camptothecin-based compound is selected from irinotecan,topotecan, belotecan, exatecan, lurtotecan, sinotecan, rubitecan,9-nitrocamptothecin, 9-am inocamptothecin, gimatecan, BNP-1530, DB-67,BN-80915, BN-80927, a pharmaceutically acceptable salt thereof, aglucuronide metabolite thereof, and a glucuronide metabolite of thehydrophobic camptothecin-based compound, but is not limited thereto.

According to a specific embodiment of the present invention, thehydrophobic camptothecin-based compound, which constitutes the particleof the present invention, may be camptothecin, SN-38, or a mixturethereof, and the hydrophilic camptothecin-based compound, whichconstitutes the particle of the present invention, may be irinotecanhydrochloride, topotecan hydrochloride, and a glucuronide analog ofSN-38.

As used herein, the term “copolymer” refers to a polymer prepared fromtwo or more different types of monomers. For example, the reaction ofstyrene acrylonitrile in a reaction container results in a copolymerhaving both of the two monomers. The term “block copolymer” refers to acopolymer having a form in which a block of one type of monomers islinked to a block of another type of monomers. A case in which a blockof material A is followed by a block of material B is expressed by-[-AB-]-. A chain is called AB type if the chain is composed of only onestrand of each monomer, ABA type if A blocks are present at both ends ofB block in the center, and ABC type if three different types of blocksare present in a main chain. A block copolymer is mainly formed by ionicpolymerization. Unlike other copolymers, this block copolymer has manyphysical properties of a homopolymer formed from two types of monomers.

According to an embodiment of the present invent, the amphiphilic blockcopolymer constituting the particle of the present invention is composedof block A-B or block A-B-A. Here, A is a hydrophilic polymer, which ismonomethoxy polyethylene glycol, dimethoxy polyethylene glycol,polyethylene glycol, polypropylene glycol, monomethoxy polypropyleneglycol, polyethylene oxide, polyacrylic acid, or a polymer thereof, butis not limited thereto. In addition, B is a hydrophobic polymer, whichis polylactic acid, polylactide, polyglycolic acid, polyglycolide, apolylactic acid-co-glycolic acid copolymer, polymandelic acid,polycaprolactone, polydioxan-2-one, polyglutamic acid, polyasparticacid, polyornithine, polyorthoester, a derivative thereof, or acopolymer of two or more compounds selected therefrom, but is notlimited thereto. It would be obvious to a person skilled in the art thatany compound that can constitute an amphiphilic block copolymer usablein the art can be used without limitation.

In a specific embodiment of the present invention, the amphiphilic blockcopolymer is PEG-PCL [poly(ethylene glycol)-b-poly(carprolactone)];PEG-PLA [poly(ethylene glycol)-b-poly(lactic acid)]; m PEG-PGA[monomethoxy poly(ethylene glycol)-b-poly(glycolic acid)]; mPEG-PLGA[monomethoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide)];PEG-PBLA [poly(ethylene glycol)-b-poly(β-benzyl-L-aspartic acid)];PEG-p(Glu) [poly(ethylene glycol)-b-poly(glutamic acid)]; PEG-p(Asp)[poly(ethylene glycol)-b-poly(aspartic acid)]; and/or PEG-PLA-PEG[poly(ethylene glycol)-b-poly(lactic acid)-b-poly(ethylene glycol).

According to an embodiment of the present invention, the hydrophilicpolymer A and the hydrophobic polymer B each has a number averagemolecular weight of 500-10,000 Da, and more specifically 1,000-7,000 Da.When the number average molecular weights of the hydrophilic polymer Aand the hydrophobic polymer B are less than 500 Da or more than 10,000Da, the produced particles have an average size of 200 nm or more andexhibit a multimodal distribution, and thus are difficult to be ananoparticle drug prescribed by the US FDA.

According to another embodiment of the present invention, the weightratio of the hydrophobic camptothecin-based compound and the hydrophiliccamptotechin-based compound, which constitute the particle of thepresent invention, is 1-10:1-10, 1-10:1-5, 1-10:1-3, 1-10:1, 1-5:1-10,1-3:1-10, or 1:1-10, specifically 1-5:1-5, 1-5:1-3, 1-5:1, 1-3:1-5, or1:1-5, and more specifically 1-3:1-3, 1-3:1, or 1:1-3, but is notlimited thereto.

According to an embodiment of the present invention the weight ratio of(a) the sum of the hydrophobic camptothecin-based compound and thehydrophilic camptotechin-based compound and (b) the amphiphilic blockcopolymer is 1:0.1-200, 1:0.5-200, 1:1-200, 1:2-200, 1:5-200, 1:10-200,1:50-200, 1:100-200, 1:150-200, 1:0.1-100, 1:0.5-100, 1:1-100, 1:2-100,1:5-100, 1:10-100, 1:20-100, 1:50-100, 1:0.1-50, 1:0.5-50, 1:1-50,1:5-50, 1:10-50, 1:20-50, 1:0.1-20, 1:0.5-20, 1:1-20, 1:5-20, 1:10-20,1:0.1-10, 1:0.5-10, or 1:1-10.

As used herein, the term “to” or “-” between two numerical values meansa section between the numerical values including numerical valuesdescribed before and after the term.

Meanwhile, the particle of the present invention has a double core-shellstructure comprising the following:

(a) an inner core-shell containing the hydrophilic camptothecin-basedcompound and the hydrophobic camptothecin-based compound; and (b) anouter core-shell containing the amphiphilic block copolymer.

In addition, the particle of the present invention has a bilayer micellestructure.

The amphiphilic block copolymer in which a hydrophilic block and ahydrophobic block are combined at a particular ratio is known to form amicelle through self-assembly in an aqueous solution. The inside of themicelle is hydrophobic, and thus the amphiphilic block copolymer isapplied as a drug delivery system for a poorly soluble preparation. Apolystyrene-polyethylene oxide double block copolymer (PS-b-PEO) is wellknown to form a spherical micelle having an insoluble core (PS) and asoluble shell (PEO) in water.

As described above, the particle of the present invention includes (a) ahydrophilic camptothecin-based compound and a hydrophobiccamptothecin-based compound; and (b) an amphiphilic block copolymer.

As proved in an example of the present invention, the mixing of thehydrophilic camptothecin-based compound and the hydrophobiccamptothecin-based compound significantly increases solubility inaqueous solvents and forms particles. Here, it is likely that thehydrophilic camptothecin-based compound serves as a hydrophilic block ofthe amphiphilic copolymer, and the hydrophobic camptothecin-basedcompound serves as a hydrophobic block of the amphiphilic copolymer,constituting a core-shell structure (micelle).

Therefore, the particle of the present invention forms a double micelle(double core-shell) containing: a monolayer micelle inside; and anamphiphilic block copolymer containing the monolayer micelle inside,wherein in the monolayer micelle, a hydrophilic camptothecin-basedcompound and a hydrophobic camptothecin-based compound constitute acore-shell structure. More specifically, the hydrophobic block of theamphiphilic block copolymer constitutes an insoluble core toward themonolayer micelle, which is relatively hydrophobic and composed ofcamptothecin-based compounds, and the hydrophilic block constitutes asoluble shell toward an external aqueous solvent, and as a result, adouble core-shell structured double micelle is formed.

According to an embodiment of the present invention, the doublecore-shell structured particles spontaneously form particles whendispersed in an aqueous solution.

According to an embodiment of the present invention, the number averageparticle size of the particles is 10-500 nm, 10-400 nm, 10-300 nm, or10-200 nm, and more specifically 20-500 nm, 20-400 nm, 20-300 nm, or20-200 nm. The number average particle size of the particles of thepresent invention shows limited change even before or after theparticles are freeze-dried. The reason seems that the hydrophobic andhydrophilic camptothecin-based compounds primarily constitute an innercore-shell structured micelle and the amphiphilic block copolymersecondarily constitutes an outer shell surrounding the micelle, so thatduring the freeze-drying, the secondary outer shell serves as acryoprotectant that prevents rapid crystallization, agglomeration,particle collapse of the primary inner core-shell. As a result, theparticles of the present invention cause no agglomeration orprecipitation even when the particles are again dissolved in the aqueoussolvent after freeze-drying. Therefore, the present invention provides astable double core-shell structured particle composition, which isadvanced from an existing a monolayer nanomicelle to resolve a problemof solubility of a severely poorly soluble drug.

According to another aspect of the present invention, the presentinvention provides a pharmaceutical composition for treating cancer, thepharmaceutical composition comprising the foregoing particles and apharmaceutically acceptable carrier.

In an embodiment of the present invention, the cancer is selected fromthe group consisting of gastric cancer, ovarian cancer, uterine cancer,small cell lung cancer, non-small cell lung cancer, pancreatic cancer,breast cancer, esophageal cancer, oral cancer, rectal cancer, coloncancer, large intestine cancer, kidney cancer, prostate cancer,melanoma, liver cancer, gall bladder, and other biliary tract cancer,thyroid cancer, bladder cancer, brain and central nervous system cancer,bone cancer, skin cancer, non-Hodgkin's and Hodgkin's lymphoma.

When the particles of the present invention or the compositioncontaining the same is prepared into a pharmaceutical composition, thepharmaceutical composition of the present invention may contain apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier is normally used at the time of formulation, and examplesthereof may include, but are not limited to, lactose, dextrose, sucrose,sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate,gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, andmineral oil. The pharmaceutical composition of the present invention mayfurther contain a lubricant, a wetting agent, a sweetening agent, aflavoring agent, an emulsifier, a suspending agent, a preservative, andthe like, in addition to the above ingredients. Suitablepharmaceutically acceptable carriers and agents are described in detailin Remington's Pharmaceutical Sciences (19^(th) ed., 1995).

In a specific embodiment of the present invention, the pharmaceuticalcomposition of the present invention may further contain sucrose,mannitol, sorbitol, glycerin, trehalose, a polyethylene glycol-basedexcipient, and a cyclodextrin-based excipient (alpha-, beta-, andgamma-cyclodextrin, hydroxy cyclodextrin, or a cyclodextrin derivative).The excipient is added to the particles, which correspond to an activeingredient of the present pharmaceutical composition, serves as acryoprotectant or an osmoregulator, and is formulated by freeze-drying,solvent evaporation, or the like.

The pharmaceutical composition of the present invention may beadministered orally or parenterally, and examples of parenteraladministration may include intravenous administration, subcutaneousadministration, intradermal administration, intramuscularadministration, intranasal administration, mucosal administration,intradural administration, intraperitoneal administration, intraocularadministration, and the like, and specifically, the pharmaceuticalcomposition of the present invention may be administered intravenously.

The suitable dose of the pharmaceutical composition of the presentinvention varies depending on factors, such as a formulating method, amanner of administration, patient's age, body weight, gender, morbidity,and food, a time of administration, a route of administration, anexcretion rate, and response sensitivity. The ordinarily skilledpractitioners can easily determine and prescribe the dose that iseffective for the desired treatment or prevention. According to anembodiment of the present invention, the daily dose of thepharmaceutical composition of the present invention is 0.001-100 mg/kg.

The pharmaceutical composition of the present invention may beformulated into a unit dosage form or may be prepared in a multi-dosecontainer by using a pharmaceutically acceptable carrier and/orexcipient according to a method that is easily conducted by a personhaving an ordinary skill in the art to which the present inventionpertains. Here, the dosage form may be a solution in an oily or aqueousmedium, a suspension, an emulsion, an extract, a powder, granules, atablet, or a capsule, and may further contain a dispersant or astabilizer.

The pharmaceutical composition of the present invention may beadministered in parallel with a known compound or pharmaceuticalcomposition having a cancer treatment effect.

In an embodiment of the present invention, the composition of thepresent invention further contains another type of anticancer drug.Specifically, the composition of the present invention further containsa poorly soluble anticancer drug, such as paclitaxel or docetaxel.

The poorly soluble anticancer drugs represented by paclitaxel anddocetaxel have low utilization due to poor solubility, like theabove-mentioned camptothecin, but the poorly soluble anticancer drugshas remarkably improved solubility when being contained in the doublemicelle particles of the present invention. Therefore, the particle ofthe present invention per se is a camptothecin-based anticancer drug,and can be favorably used as a drug delivery system platform, which canload poorly soluble anticancer drugs or novel candidate drugs with lowsolubility problems to improve solubility thereof.

According to still another aspect of the present invention, the presentinvention provides a method for cancer treatment, the method comprisingadministering the foregoing pharmaceutical composition of the presentinvention to a subject.

As used herein, the term “administration” or “administer” refers to thedirect application of a therapeutically effective amount of thecomposition of the present invention to a subject (i.e., an object) withcancer, thereby forming the same amount thereof in the body of thesubject.

The term “therapeutically effective amount” of the composition refers tothe content of the composition, which is sufficient to provide atherapeutic or preventive effect to a subject to be administered, andthus the term has a meaning including “prophylactically effectiveamount.” As used herein, the term “subject” includes, but is not limitedto, human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey,chimpanzee, baboon, or rhesus monkey. Specifically, the subject of thepresent invention is human.

Since the method for cancer treatment of the present invention includesa step of administering the pharmaceutical composition for cancertreatment according to an aspect of the present invention, theoverlapping descriptions therebetween are omitted to avoid excessivecomplication of the specification.

According to another aspect of the present invention, there is provideda method for manufacturing a particle, the method comprising:

(a) forming an inner core-shell containing a hydrophobiccamptothecin-based compound and a hydrophilic camptothecin-basedcompound; and

(b) forming an outer core-shell containing an amphiphilic copolymer.

In an embodiment of the present invention, step (a) comprises mixing thehydrophobic camptothecin-based compound and a hydrophiliccamptothecin-based compound in an organic solvent; and step (b)comprises mixing the inner core-shell and the amphiphilic blockcopolymer in an aqueous solvent.

Since the hydrophobic camptothecin-based compound of the presentinvention is severely hydrophobic, a particular solubilizing techniqueor preparation method is needed. In an example of the present invention,in order to overcome such poor solubility, a hydrophobiccamptothecin-based compound and a hydrophilic camptothecin-basedcompound were, primarily, simultaneously dissolved in an organicsolvent, and then an organic solvent was completely removed by utilizinga rotary vacuum evaporator, thereby obtaining a film form mixture ofcamptothecin-based compounds. Here, a small amount of aqueous solvent(e.g., distilled water) was added thereto with intensive mixing using avortex-mixer or with ultrasonication, thereby obtaining a nano-sizedprimary core-shell complex.

Then, an amphiphilic polymer previously dissolved in an aqueous solvent(e.g., distilled water) was added to the primary core-shell complex,followed by homogeneous and strong mixing using a vortex-mixer orultrasonication, thereby obtaining nano-sized double core-shellparticles. Finally, a cryoprotectant and an isotonizing agent was addedthereto to be completely dissolved therein, and then the resultantsolution was filtered through a 0.22-μm sterile filter, followed byfreeze-drying. When added to a 0.9% sodium chloride injection, a 5%glucose injection, or injection water, the final freeze-dried materialis self-assembled to form double core-shell particles with a numberaverage particle size of 20-200 nm.

In the case where the particles manufactured according to the presentinvention have a particle size of 200 nm or less, the non-selectiveremoval of a reticuloendothelial (RES) system in the body can be avoid,and thus it is preferable to manufacture particles having a uniformparticle size of 200 nm or less.

In another embodiment of the present invention, step (a) comprisesmixing a basic aqueous solution, in which a hydrophobic camptothecincompound is dissolved, and an aqueous solution, in which a hydrophiliccamptothecin compound is dissolved; and step (b) comprises mixing theinner core-shell and the amphiphilic block copolymer in an aqueoussolvent.

Here, the aqueous solution in which the hydrophilic camptothecincompound is dissolved may be a basic, neutral, or acidic aqueoussolution.

The primary inner core-shell can be manufactured by the following methodbesides the manufacturing method in an organic solvent. The hydrophobiccamptothecin-based compound (e.g., camptothecin, SN-38) has very lowsolubility in an acidic or neutral aqueous solution of pH 7 or less, butthe solubility of the hydrophobic camptothecin-based compound is rapidlyincreased in a basic aqueous solution since a lactone ring is opened tohave a form of carboxylic acid. Therefore, the hydrophobiccamptothecin-based compound is dissolved in the basic aqueous solution,and then an aqueous solution in which a hydrophilic camptothecin-basedcompound is dissolved is added thereto to lower the pH to 7 or less, sothat the hydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound form a micelle, and the acidity isneutralized and the lactone ring is closed, thereby restoring to theoriginal chemical structure thereof.

In a specific embodiment of the present invention, step (a) comprises:(a1) mixing a basic aqueous solution, in which a hydrophobiccamptothecin-based compound is dissolved, and a basic, neutral, oracidic aqueous solution, in which a hydrophilic camptothecin-basedcompound is dissolved; and (a2) lowering the pH of the mixed aqueoussolution to 7 or lower.

In an example of the present invention, camptothecin or SN-38 wasdissolved in a basic aqueous solution, and a hydrophiliccamptothecin-based compound previously dissolved in an aqueous solutionwas added to the basic aqueous solution with strong mixing usingultrasonication or a vortex-mixer, and an acidic aqueous solution wasfurther added thereto to adjust the pH to 7 or less, thereby obtainingprimary core-shell particles. An amphiphilic polymer previouslydissolved in an aqueous solvent (e.g., distilled water) was added to amixture of the produced primary core-shell particles throughvortex-mixing or ultrasonication, thereby manufacturing a doublecore-shell mixture. A cryoprotectant and an isotonizing agent wasfurther added thereto to be dissolved therein, followed by sterilefiltration using a 0.22-μm filter, and followed by freeze-drying.

In the manufacturing method of the present invention, the acidic aqueoussolution contains a pharmaceutically acceptable inorganic acid, such ashydrochloric acid, nitric acid, sulfuric acid, or phosphoric acid, andat least one organic acid selected from the group consisting of citricacid, malic acid, lactic acid, acetic acid, and tartaric acid. The pH ofthe acidic aqueous solution is 1-6. In addition, the manufacturingmethod of the present invention, the basic aqueous solution contains: aninorganic alkali, including sodium hydroxide, potassium hydroxide,sodium dihydrogenphosphate, potassium dihydrogenphosphate, magnesiumhydroxide, sodium carbonate, and sodium hydrogencarbonate; an alkalisalt of an organic acid; an alkyl amine; or a mixture thereof. The pH ofthe basic aqueous solution is 8-13.

In still another embodiment of the present invention, step (a) includesmixing the hydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound in an organic solvent; and step (b) includesmixing a mixture of the hydrophobic camptothecin-based compound and thehydrophilic camptothecin-based compound with an amphiphilic blockcopolymer in an organic solvent.

In the present invention, step (a) (manufacturing of inner core-shell)and step (b) (manufacturing of external core-shell) can be performed inone step.

An example of the present invention showed a method for simultaneouslymanufacturing primary inner core-shell and secondary outer core-shellparticles. A hydrophobic camptothecin (e.g., 10-hydroxycamptothecin orSN-38), together with a hydrophilic camptothecin (e.g., irinotecanhydrochloride), was added into an organic solvent, and completelydissolved with stirring, and an amphiphilic block copolymer (e.g.,mPEG-PLA) previously dissolved in an organic solvent was added theretowith stirring. The mixture solution was dried by a rotary vacuumevaporator, and an aqueous solvent (e.g., distilled water) was added toresidues, followed by ultrasonication in an ultrasonic cleaner for 10minutes, thereby manufacturing double nanomicelle particles.

In the manufacturing method of particles of the present invention, thedefinitions of a hydrophobic camptothecin-based compound, a hydrophiliccamptothecin-based compound, and an amphiphilic block copolymer, whichconstitute the particles, are as described above with respect to theparticles of the present invention.

In the manufacturing method of the present invention, the organicsolvent is a C1-05 alcohol (methanol, ethanol, propanol, butanol,n-butanol, iso-propanol, 1-pentanol, 2-butoxyethanol, isobutyl alcohol,etc.), an alkyl acetate, acetone, an acetonitrile, chloroform, benzene,toluene, xylene, acetone, a fluoroalkane, pentane, hexane,2,2,4-trimethyl pentane, a decane, a cyclohexane, diisobutylene,1-pentene, 1-chlorobutane, 1-chloropentane, diisopropyl ether,2-chloropropane, 1-chloropropane, chlorobenzene, benzene, diethyl ether,diethyl sulfide, dichloromethane, 1,2-dichloroethane, an aniline, adiethyl amine, an ether, carbon tetrachloride, tetrahydrofuran (THF), ora mixed solvent thereof, but is not limited thereto.

Advantageous Effects

The double core-shell structured particles manufactured by the presentinvention do not precipitate into crystals even when diluted to asufficiently low concentration in an aqueous solution, leading to verystable particles. The particles of the present invention show amono-distribution of particles in an injection solvent before and afterfreeze-drying. The proportion of particles of 200 nm or more is 10% orless, and particles of 500 nm or more are not present. Furthermore, thepresent invention shows excellent results, compared with existingmonolayer micelles in animal efficacy tests and pharmacokinetic tests,and does not use a surfactant (Cremophore EL, Pluronic, etc.) causinghypersensitivity, and thus the use of the particles of the presentinvention can provide a pharmaceutical composition or a drug deliverysystem platform, which are safe for the human body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b are graphs showing the dynamic light scattering (DLS)measurement results of the sizes of particles formed in an aqueoussolution after freeze-drying, with respect to core-shell particles(monolayer micelles) composed of SN-38 and irinotecan according to anexample of the present invention. FIG. 1c is a graph showing the dynamiclight scattering (DLS) measurement results of the sizes of particlesformed in an aqueous solution after freeze-drying, with respect todouble core-shell particles (bilayer micelle) according to an example ofthe present invention.

FIGS. 2 and 3 are a graph and images of extracted tumors, respectively,for comparing a tumor inhibitory effect of core-shell particles(monolayer micelles) and double core-shell particles (bilayer micelles)in colorectal cancer mouse models according to an example of the presentinvention.

FIG. 4 is a graph for comparing a tumor inhibitory effect betweencore-shell particles (monolayer micelles) and double core-shellparticles (bilayer micelles) in pancreatic cancer mouse models (AsPc-1,Xenograft) according to an example of the present invention.

FIGS. 5a and 5b are a graph and images of extracted tumors,respectively, for comparing a tumor inhibitory effect of core-shellparticles (monolayer micelles) and double core-shell particles (bilayermicelles) in pancreatic cancer mouse models (MiaPaca-2, Orthotopic)according to an example of the present invention.

FIG. 6 is a graph showing the blood concentration of SN-38 Glucuronidefor comparing pharmacokinetic characteristics between core-shellparticles (monolayer micelles) and double core-shell particles (bilayermicelles) of the present invention.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail withreference to examples. These examples are only for illustrating thepresent invention more specifically, and it will be apparent to thoseskilled in the art that the scope of the present invention is notlimited by these examples.

EXAMPLES

Throughout the present specification, the term “%” used to express theconcentration of a specific material, unless otherwise particularlystated, refers to (wt/wt)% for solid/solid, (wt/vol)% for solid/liquid,and (vol/vol)% for liquid/liquid.

Materials

Among the compounds used in the present invention, hydrophobic andhydrophilic camptothecin-based compounds, polysaccharides such astrehalose, cyclodextrin, polyethylene glycol, and the like were usedfrom Sigma-Aldrich, AbCem, Toronto Research Chemical (Canada) or Tocris(USA), and polymers were used from Akina Inc (USA), Advanced PolymerMaterials (Canada), Shanghai Liang Chemical Co., LTD (China), NanoSoftPolymer (USA), and Samyang BioPharm (Korea).

Example 1 Solubility in Different Solvents, when HydrophobicCamptothecin Compound was Dissolved Alone or Hydrophobic CamptothecinCompound and Hydrophilic Camptothecin Compound were Dissolved in Mixture

The solubility change according to solvent was measured when thehydrophobic camptothecin compound, camptothecin or7-ethyl-10-hydroxy-camptothecin (SN-38) was dissolved alone or togetherwith the hydrophilic camptothecin-based compound, irinotecan (CPT-11),topotecan, or belotecan (CKD-602).

1) Solubility in Different Solvents when Hydrophobic CamptothecinCompound was Dissolved Alone

A supersaturated solution was prepared by adding 20 mg of thehydrophobic camptothecin compound (camptothecin or SN-38) to 5 ml ofethanol, acetonitrile, acetone, ethyl acetate, chloroform dimethylsulfoxide, or distilled water, followed by ultrasonic treatment for 30minutes. The prepared supersaturated solution was filtered through a0.45-μm filter, and a filtrate was properly diluted, followed by HPLCanalysis.

2) Solubility in Different Solvents when Hydrophobic CamptothecinCompound and Hydrophilic Cam Ptothecin Compound were Dissolved inMixture

The present inventors prepared a supersaturated solution by adding 20 mgof the hydrophilic camptothecin compound (irinotecan hydrochloride,topotecan hydrochloride, or belotecan) to 5 ml of ethanol, acetonitrile,chloroform, ethyl acetate, dimethyl sulfoxide, or distilled water, andthen adding 20 mg of the hydrophobic camptothecin compound (SN-38 orcamptothecin), followed by ultrasonication for 30 minutes. The preparedsupersaturated solution was filtered through a 0.45-μm filter, and afiltrate was properly diluted, followed by HPLC analysis.

HPLC (Agilent 1200 Series, USA) conditions were as follows. The columnwas CapcellPak C8 (5 m, 4.6 mm×25 cm, Shiseido); the mobile phase was amixed solvent of methanol:acetonitrile:buffer (2.8 g/L sodiumdihydrogenphosphate, 1.8 g/L 1-octanesulfonate aqueoussolution)=17:24:59 (v/v); the flow rate was 1.5 mL/min; the measurementwavelength was UV 255 nm; and the sample injection amount was 15 μL. Thesolubility test results are shown in table 1 below.

TABLE 1 Solubility changes of hydrophobic camptothecin-based compoundsin polar organic solvents (unit: mg/ml) Comparative Test ComparativeTest Test Test Solvent type example example example example exampleexample Camptothecin Camptothecin SN-38 SN-38 SN-38 SN-38 20 mg 20 mg 20mg 20 mg 20 mg 20 mg — Irinotecan Irinotecan Topotecan Belotecan 20 mg20 mg 20 mg 20 mg Ethanol 0.149 1.414 0.779 2.885 2.205 2.047Acetonitrile 0.101 1.090 0.402 1.972 1.694 1.338 Chloroform 0.002 0.0510.009 0.025 0.088 0.104 Ethyl 0.134 0.295 0.078 0.425 0.239 0.221acetate Dimethyl 3.291 >4 2.263 3.816 2.949 2.685 sulfoxide Distilled0.009 0.011 <0.001 <0.001 <0.001 <0.001 water

As shown in Table 1 above, the poorly soluble compound camptothecin orSN-38 alone was hardly dissolved in most solvents including water, andwere dissolved at 2.26-3.29 mg/mL in only dimethyl sulfoxide (DMSO) as anon-volatile solvent. However, camptothecin or SN-38, when dissolvedtogether with the relatively hydrophilic drug, irinotecan hydrochloride,topotecan hydrochloride, or belotecan, had solubility increased up to15-fold, which corresponds to an appropriate level of solubilityrequired for the manufacture of drugs. It was therefore confirmed fromthe above results that the solubility of the hydrophobic camptothecincompounds was remarkably increased when dissolved in mixing with ahydrophilic camptothecin compound.

Example 2 Manufacturing of Primary Core-Shell (monolayer Micelles) andSecondary Core-Shell (Double Core-Shell Particles) from HydrophobicCamptothecin-Based Compound and Hydrophilic Camptothecin-Based CompoundUnder Organic Solvent and Evaluation of Particle Size

In the present example, the hydrophobic camptothecin-based compoundcamptothecin or SN-38, and the hydrophilic camptothecin-based compoundirinotecan hydrochloride, topotecan hydrochloride, belotecan, orSN-38-glucuronide, or an amphiphilic polymer (PEG-PBLA) were dissolvedin an organic solvent to manufacture primary core-shell particles(monolayer micelles), and an amphiphilic polymer was added thereto tomanufacture secondary core-shell particles (bilayer micelles).

1) Manufacturing of Primary Core-Shell (monolayer Micelles) (ComparativeExamples 1 to 6)

The primary core-shell particles as monolayer micelles were manufacturedby using, as a main ingredient, a hydrophobic camptothecin (camptothecinor SN-38), and water-soluble camptothecin (irinotecan hydrochloride,topotecan hydrochloride, or SN-38-glucuronide) as an amphiphiliccompound for micelle formation or an amphiphilic polymer(poly(ethyleneglycol)-poly (β-butyrolactone-co-lactic acid); PEG-PBLA),as shown in table 2 below.

Specifically, 20 mg of water-soluble camptothecin (irinotecanhydrochloride, topotecan hydrochloride, or SN-38 glucuronide) orPEG-PBLA was added to 20 mg of hydrophobic camptothecin, and 100 ml ofan organic solvent (a 50:50 mixture solution of ethanol andacetonitrile) was added thereto to attain complete dissolution, followedby drying in a rotary vacuum evaporator. 20 ml of distilled water wasadded to the dried product, followed by ultrasonication at 20-30° C. for20 minutes in an ultrasonic cleaner (UC-20, 20Hz, 400W, Jeio Tech,Korea), thereby obtaining nano-sized particles (monolayer micelles) in adispersed state in an aqueous solution. 600 mg of D-trehalose was addedto the aqueous solution containing the nano-sized particles, therebyattaining complete dissolution, and then the solution was filteredthrough a 0.22 μm sterile filter, and the filtrate was freeze-dried. Thefreeze-drying was conducted for a total of 62 hours under a temperaturecycle of −45° C.→−20° C.→0° C.→20° C. at a vacuum pressure of 100 mTorror lower, and a freeze-drier from Operon (Korea) was used. An aliquot ofthe prepared freeze-dried product was taken, and again dissolved indistilled water for injection, and the size of particles was measured bythe dynamic light scattering (DLS) (Zetasizer™, Malvern, UK).

2) Manufacturing of Double Core-Shell (Bilayer Micelles) (Test Examples1 to 4)

In order to prepare a bilayer particle composition having a doublecore-shell structure, the present inventors added 20 mg of each ofhydrophobic cam ptothecin and hydrophilic camptothecin into 100 ml of anorganic solvent (a 50:50 mixture solution of ethanol and acetonitrile)to be dissolved therein, followed by drying using a rotary vacuumevaporator (Buchi). 200 ml of distilled water was added to the driedproduct, followed by ultrasonication at 20-30° C. for 20 minutes in anultrasonic cleaner, thereby obtaining nano-sized core-shell particles(monolayer micelles) in a dispersed state in an aqueous solution. Whilethe aqueous solution mixed with the core-shell particles was stirred, 90mg of the amphiphilic block copolymer methoxy poly(ethyleneglycol)-poly(lactide) (mPEG-PLA) (mPEG molecular weight:PLA molecularweight=2,000:1,500) previously dissolved in 10 ml of distilled water wasslowly added, followed by stirring at 20-30° C. for 6 hours, therebypreparing double core-shell particles in a dispersed state in an aqueoussolution. 600 mg of D-trehalose as a cryoprotectant was added to theaqueous solution containing double core-shell particles to be dissolvedtherein, and then the mixture was filtered through a 0.22-μm sterilefilter, and then freeze-drying was conducted by the same method as inthe manufacturing of primary core-shell particles, thereby obtaining afreeze-dried product as a white powder. A predetermined amount of thefreeze-dried product was again dissolved in injection water to measurethe size of the particles. In addition, the average particle sizesafter/before freeze-drying were measured, and the proportion of particlewith 200 nm or more and the distribution form (mono- or multi-modaldistribution) were compared.

TABLE 2 Proportions in manufacturing of monolayer micelles and bilayerparticles (unit: mg) Poorly soluble camptothecin Water-solublecamptothecin PEG- mPEG- Micelle SN- Irinotecan Topotecan SN-38 PBLA PLAtype Camptothecin 38 hydrochloride hydrochloride glucuronide (5k:6.5k)(2k:1.5k) Comparative Mono- 20 — 20 — — — — example 1 Comparative Mono-20 — — — 20 — — example 2 Test Bi- 20 — 20 — — — 90 example 1Comparative Mono- — 20 20 — — — — example 3 Comparative Mono- — 20 — 20— — — example 4 Comparative Mono- — 20 — — 20 — — example 5 ComparativeMono- — 20 — — — 20 — example 6 Test Bi- — 20 20 — — — 90 example 2 TestBi- — 20 — 20 — — 90 example 3 Test Bi- — 20 — — 20 — 90 example 4*PEG-PBLA: poly(ethylene glycol)-b-poly(β-benzyl-L-aspartic acid)*mPEG-PLA: methoxypoly(ethylene glycol)-b-poly(lactic acid)

TABLE 3 Comparision results of particle size before/after freeze- dryingin monolayer micelles and bilayer particles Particle size before freeze-Particle size after freeze- drying (n = 3) drying (n = 3) Average >200nm Average >200 nm Micelle particle size Propotion particle sizeproportion Micelle type (nm) (%) (nm) (%) >1 μm distribution ComparativeMono- 123.9 5.9 156.1 40.1 ◯ Multi- example 1 Comparative Mono- 133.67.1 152.8 38.6 ◯ Multi- example 2 Test example 1 Bi- 135.8 3.6 148.9 4.8ND Mono- Comparative Mono- 76.2 0.0 108.4 16.4 ◯ Multi- example 3Comparative Mono- 78.5 0.1 115.4 28.5 ◯ Multi- example 4 ComparativeMono- 92.1 2.7 131.2 30.4 ◯ Multi- example 5 Comparative Mono- 102.8 5.9138.4 34.2 ◯ Multi- example 6 Test example 2 Bi- 83.3 0.0 93.8 2.4 NDMono- Test example 3 Bi- 88.2 0.1 99.8 2.8 ND Mono- Test example 4 Bi-93.3 0.2 105.4 3.1 ND Mono- *ND: Not detected

As shown in Table 3 above, as for the average particle size afterfreeze-drying, the primary core-shell particles (monolayer micelles)composed of poorly soluble camptothecin and water-soluble camptothecinor the primary core-shell particles composed of poorly solublecamptothecin and an amphiphilic polymer were increased to about 1.2- to1.5-fold, but the double-core-shell particles (bilayer micelles) onlyincreased about 1.1-fold.

With respect to the primary core-shell particles (monolayer micelles),after the freeze-drying, the particles of 200 nm or more were producedin large amounts, about 16-40%, and especially, the particles of severalmicrometers (μm) or more, capable of influencing safety whenadministered to the human body, were produced (comparative examples 1 to6 on table 3 and FIGS. 1a and 1b ). Whereas, with respect to doublecore-shell particles, the particles of 200 nm or more were detected inabout 2.4-3.1% for SN-38 and about 4.8% for camptothecin, both beingless than 5%, showing very favorable results, and the particles of 500nm or more were not observed (Test examples 1 to 4 in table 3, and FIG.1c ).

In the particular distribution, the primary core-shell particles(monolayer micelles) showed a distribution with multiple peaks (FIGS. 1aand 1b ), but the secondary core-shell particles showed amono-distribution, confirming a very stable structure (FIG. 1c ). Inaddition, the average particle size of the primary core-shell particles(monolayer micelles) was smaller than that of the double core-shellparticles (bilayer micelles) by about 10 nm before freeze-drying, butthe change of the particle size before and after freeze-drying was verygreat, with the result that the double core-shell particles (bilayermicelles) were very physically stable.

Example 3 Evaluation of Stability of Primary Core-Shell Particles(Monolayer Micelles) and Double Core-Shell Particles (Bilayer Micelles)

In the present example, monolayer micelles (comparative examples 1, 3,and 6) and double core-shell particles (test examples 1 and 2) werecompared for the change in particle size, wherein sample productsmanufactured according to the drug stability test standards were storedfor six months in accelerated test conditions (40° C., 75% relativehumidity). The results are shown in Table 4.

TABLE 4 Test results of stability of primary core-shell particles(monolayer micelles) and double core-shell particles (bilayer micelles)0 month 3 months 6 months Average >200 nm Average >200 nm Average >200nm Micelle particle size Propotion particle size Propotion particle sizePropotion type (nm) (%) (nm) (%) (nm) (%) Comparative Mono- 156.1 40.1172.1 47.6 196.6 58.1 example 1 Test example 1 Bi- 148.9 4.8 155.0 5.6168.3 7.4 Comparative Mono- 108.4 16.4 126.7 28.1 151.9 31.6 example 3Comparative Mono- 138.4 34.2 149.9 44.6 174.2 48.4 example 6 Testexample 2 Bi- 93.8 2.4 99.5 3.5 112.9 3.8

The primary core-shell particles (monolayer micelles), containingcamptothecin or SN-38 as a main ingredient, and double core-shellparticles (bilayer micelles) were subjected to stability tests. As aresult, in the case of the monolayer micelles, the average particle sizewas increased by about 50% or more and the proportion of the particlesof 200 nm or more was rapidly increased to 58% in accelerated testconditions. In the case of the bilayer particles, the average particlesize was increased by about 20%, and the proportion of the particles of200 nm or more was restricted within 3.8% for SN-38 and 7.4% forcamptothecin. Therefore, it can be seen that the structure of thebilayer particles of the present invention was significantly improved inview of stability, compared with the monolayer micelles.

Example 5 Manufacturing of Double Core-Shell Particles According to Typeof Amphiphilic Polymer and Molecular Weight of Amphiphilic Polymer

In the present example, double core-shell particles were manufacturedusing various amphiphilic polymers (block copolymers). As shown in Table5, 20 mg of each of SN-38 and irinotecan hydrochloride as hydrophobicand hydrophilic cam ptothecin compounds, 500 mg of trehalose as acryoprotectant, and 90 mg of each of amphiphilic polymers were used. Themanufacturing method was carried out in the same manner as in thecore-shell particle manufacturing method in example 2 above, and thematerials were again dissolved in injection water after freeze-drying tocompare particular sizes.

TABLE 5 Particle size comparision of bilayer particles afterfreeze-drying according to amphiphilic polymer type and averagemolecular weight thereof Polymer type Particle >200 nm >1 μm Particle(Average molecular size Proportion presence distribution No weight) (nm)(%) or absence (Mono-/Multi-) PDI Test example 5 PEG-PCL (5k:2.5k) 133.85.0 ND Mono- 0.271 Test example 6 PEG-PCL(2k:1.5k) 102.0 3.1 ND Mono-0.209 Test example 7 PEG-PLA(2.5k:1k) 99.2 2.9 ND Mono- 0.225 Testexample 8 mPEG-PGA(2k:1.5k) 99.7 2.6 ND Mono- 0.213 Test example 9mPEG-PLGA(1k:1k) 101.5 3.4 ND Mono- 0.247 Test example 10PEG-PBLA(5k:6.5k) 137.9 9.8 ND Mono- 0.287 Test example 11PEG-p(Glu)(5k:2.5k) 122.1 6.5 ND Mono- 0.245 Test example 12mPEG-p(Asp)(5k:2.5k) 128.2 7.6 ND Mono- 0.231 Test example 13PEG-PLA-PEG 156.4 11.8 ND Mono- 0.298 (2.5k-1k-2.5k) *PEG-PCL:poly(ethylene glycol)-b-poly(caprolactone) *PEG-PLA: poly(ethyleneglycol)-b-poly(lactic acid) *mPEG-PGA: monomethoxy poly(ethyleneglycol)-b-poly(glycolic acid) *mPEG-PLGA: monomethoxy poly(ethyleneglycol)-b-poly(lactide-co-glycolide) *PEG-PBLA: poly(ethyleneglycol)-b-poly(β-benzyl-L-aspartic acid) *PEG-p(Glu): poly(ethyleneglycol)-b-poly(glutamic acid) *PEG-p(Asp): poly(ethyleneglycol)-b-poly(aspartic acid) *PEG-PLA-PEG: poly(ethyleneglycol)-b-poly(lactic acid)-b-poly(ethylene glycol) *PDI: Polydiversityindex

As shown in Table 5 above, it can be seen that the bilayer particles ofthe present invention can be manufactured by using various amphiphilicpolymers (block copolymers) and amphiphilic polymers having variousaverage molecular weights, and the stability of the particles wasexcellent.

Example 6 Evaluation of Particle Size According to Type ofCryoprotectant

In the present example, the effects of a cryoprotectant in themanufacturing of primary core-shell particles (monolayer micelles) anddouble core-shell particles were observed. Here, 10-hydroxycamptothecinand SN-48 were selected as hydrophobic camptothecin compounds;irinotecan hydrochloride was selected as a hydrophilic camptothecin; andmPEG-PLA (2 k:1.5 k) was used as an amphiphilic polymer. As acryoprotectant, 500 mg of each of D-trehalose, D-mannitol, PEG2000, andhydroxypropyl-β-cyclodextrin (HP-b-CD) was used. The double core-shellparticles were manufactured using the compositions shown in Table 6, andthe manufacturing method was carried out in the same manner as inexample 2.

TABLE 6 Size of double core-shell particles according to cryoprotectanttype 10- Cryo- Particle >200 nm >1 μm OHCamptothecin SN-38 IrinotecanmPEG- protectant size Proporiton presence or (mg) (mg) (mg) PLA (500 mg)(nm) (%) absence Test example 13 20 — 20 90 Mannitol 121.8 4.4 ND Testexample 14 — 20 20 90 Mannitol 101.1 2.9 ND Test example 15 20 — 20 90Trehalose 118.6 5.8 ND Test example 16 — 20 20 90 Trehalose 99.6 3.1 NDTest example 17 — 20 20 90 PEG2000 133.2 19.6 ◯ Test example 18 — 20 20— PEG2000 164.9 32.8 ◯ Test example 19 — 20 20 90 HP-b-CD 126.8 9.3 ◯Test example 20 — 20 20 — HP-b-CD 151.7 26.2 ◯ *PEG2000:Polyethyleneglycol 2000 *HP-b-CD: hydroxypropyl-β-cyclodextrin *ND: Notdetected

As shown in Table 6, favorable results were observed in view of theparticle size when the polysaccharides mannitol and trehalose were usedas cryoprotectants. Whereas, the particle sizes were somewhat large, forexample, particles with a size of 1 μm or more were detected, in PEG2000and HP-b-CD. In test examples 18 and 20 for monolayer micelles,relatively large particle of 200 nm or more and macroparticles of 1 μmor more were observed when polyethylene glycol and cyclodextrin wereused as cryoprotectants.

Example 7 Manufacturing of Primary Core-Shell Particles and DoubleCore-Shell Particles in Water-Soluble Solvent Conditions

The primary core-shell particles (monolayer micelles) can bemanufactured in an aqueous solution as well as an organic solvent as inexample 2. As shown in Table 7 below, 10 mg of SN-38 was completelydissolved in 0.1 ml of a 0.5 M sodium hydroxide aqueous solution, andthe resultant solution was dropped and neutralized in an aqueoussolution of irinotecan hydrochloride (1 mg/ml) previously dissolved in15 ml of a 0.5 mM hydrochloride aqueous solution, and a hydrochlorideaqueous solution was further added to control the pH to about 5,followed by ultrasonication, thereby obtaining primary core-shellparticles (monolayer micelles). 40 mg of the amphiphilic polymermPEG-PLA was added thereto, followed by stirring at room temperature for6 hours, and 300 mg of D-trehalose was further added to be dissolved.The mixture solution was filtered through a 0.22-μm sterile filter,freeze-dried, and again dissolved in injection water, and then theparticle size was measured (test example 21). SN-38 and irinotecan weredissolved using the organic alkali ethanol amine as a basic aqueoussolution or the organic acid citric acid as an acidic aqueous solution,and bilayer particles were manufactured by the same method, therebymeasuring the particle sizes, respectively (test examples 22 and 23).

TABLE 7 Manufacturing of bilayer particles under basic and acidicaqueous solutions Type of acid and basic solvents and particle size (nm)SN-38 Irinotecan mPEG-PLA NaOH/ NaOH/ (mg) (mg) Trehalose (2k:1.5k) HClEthanolamine/HCl Citric acid Test example 21 10 10 300 40 120.1 — — Testexample 22 10 10 300 40 — 122. 3 — Test example 23 10 10 300 40 — —119.5

As shown in Table 7, the double core-shell particles manufactured bydissolving hydrophobic camptothecin and hydrophilic camptothecin inbasic and acidic aqueous solutions showed a particle size of about 120nm, and thus the double core-shell particles were successfullymanufactured.

Example 8 Mixing Manufacturing of Double Core-Shell Particles

The present example showed a method for manufacturing double core-shellparticles by mixing all the hydrophobic camptothecin, hydrophiliccamptothecin, and amphiphilic block copolymer in one step. Hydrophobiccamptothecin compounds (10-hydroxycamptothecin and SN-38) were placedtogether with 20 mg of irinotecan hydrochloride in 100 ml of an organicsolvent (50:50 mixture solution of ethanol:acetonitrile), and completelydissolved with stirring, and 90 mg of mPEG-PLA (2 k:1.5 k) dissolved in10 ml of an organic solvent (50:50 mixture solution ofethanol:acetonitrile) was added thereto with stirring. The mixturesolution was dried by a rotary vacuum evaporator, and 200 ml ofdistilled water was added to residues, followed by ultrasonication for10 minutes in an a ultrasonic cleaner, thereby obtaining doublecore-shell particles of the present invention. 400 mg of D-mannitol as acryoprotectant was added to be dissolved, and this solution was filteredthrough a 0.22-μm sterile filter, freeze-dried. A proper amount of thefreeze-dried product was again dissolved in injection water to measurethe particle size. The results are shown in Table 8.

TABLE 8 Particle size of double core-shell particles (bilayer micelles)produced by mixing manufacturing SN- 10-OH mPEG- D- Particle >200 nm >1μm 38 Camptothecin Irinotecan PLA mannitol size Proportion Presence or(mg) (mg) (mg) (mg) (mg) (nm) (%) absence Test 20 — 20 90 400 125.4 4.2ND example 24 Test — 20 20 90 400 129.3 4.8 ND example 25 *ND: Notdetected

As shown in Table 8, the double core-shell particles manufactured bysimultaneously mixing and dissolving hydrophobic camptothecin,hydrophilic camptothecin, and amphiphilic polymer in an organic solventshowed a mono-distribution of particle sizes of about 120-130 nm,wherein particles of 200 nm or more were detected in small amounts, 5%or less, but particles of 1 μm or more were not detected, and thus theparticles were confirmed to have overall favorable stability.

Example 9 Tumor Inhibitory Effect Comparison Test of Primary Core-ShellParticles (Monolayer Micelles) and Double Core-Shell Particles (BilayerMicelles) in Tumor Mouse Models (Colorectal Cancer)

In colorectal mouse models, monolayer micelle compositions (comparativeexamples 3 and 6) and bilayer particle composition (test example 2) weremeasured for anticancer effect by the following method.

The previously cultured colorectal cancer cell line (HT-29) was injectedat 5×10⁶ cells/0.2 mL into the right flank of Balb/c nude mice, andafter about 7 days, only tumors with a size of 150-200 mm³ wereselected. Nine animals were assigned to each group, and wereintravenously administered with a sham drug (non-treatment group),comparative example (monolayer micelles), comparative example 6(monolayer micelles), and test example 2 (bilayer particles), once everythree days, three times in total. The dose was 10 mg/kg on the basis ofSN-38. The volume of tumor measured every three days after theadministration of the test composition was used as a measurement indexof the anticancer effect, and was observed for a total of 18 days. Theresults are shown in FIGS. 2 and 3.

As a result of measurement of tumor inhibitory effect, the monolayermicelle compositions (comparative examples 3 and 6) showed a tumorinhibitory effect of about 50-60% compared with a negative controlgroup, and the bilayer particle composition (test example 2) showed atumor inhibitory effect of about 80% or more compared with a negativecontrol group, indicating very excellent effects. These results were dueto the fact that the stabilized micelle structure and the micellestructure having a size as small as 200 nm or less of the presentinvention were efficiently transferred to cancer tissues while stablystaying in the body.

Example 10 Tumor Inhibitory Effect Comparison Test of Monolayer Micelleand Bilayer Particles in Pancreatic Cancer Mouse Models (AsPc-1)

The tumor inhibitory effect of the monolayer micelle composition(comparative example 3) was compared with that of the bilayer particlecomposition (test example 2) in pancreatic cancer mouse models. Thepreviously cultured pancreatic cancer cell line (AsPc-1) was injected at5×10⁶ cells/0.2 mL into the right flank of male BALB/c-nu/nu mice, andafter about 10 days, only tumors with a size of 100-150 mm³ wereselected. Ten animals were assigned to each group, and were administeredwith a sham drug (non-treatment group), comparative example 3, and testexample 2, once every seven days, three times in total. The dose was 10mg/kg on the basis of SN-38. The volume of tumor measured every threedays after the administration of the test composition was used as ameasurement index of the anticancer effect, and was observed for a totalof 24 days. The results are shown FIG. 4.

As a result of measurement of tumor inhibitory effect, the monolayermicelle composition (comparative example 3) showed a tumor inhibitoryeffect of about 27% compared with a negative control group, and thebilayer particle composition (test example 2) showed a tumor inhibitoryeffect of about 47% or more compared with a negative control group,indicating very excellent effects. The results were overall similar tothose in the colorectal cancer models (Example 9), and it was confirmedthat the bilayer particle composition of the present invention were veryexcellent in tumor inhibitory effects compared with monolayer micelles.

Example 11 Tumor Inhibitory Effect Comparison Test of Monolayer Micelleand Bilayer Particle Composition in Pancreatic Cancer Mouse Models(MiaPaca-2)

The tumor inhibitory effect of the monolayer micelle composition(comparative example 3) was compared with that of the bilayer particlecomposition (test example 2) in pancreatic cancer mouse models(Orthotopic) on the basis of SN-38. After the left flank side of maleBALB/c-nu/nu mice was incised at 0.7-1 cm, the entire pancreas andspleen were exposed to the outside, and then the pancreatic cancer cellline (MiaPaca-2 cell line) previously cultured using a syringe wasinjected at 1×10⁷ cells/0.1 mL. It was confirmed that the tumor cellsuspension did not leak, and the organs exposed to the outside wererelocated again, and the incision site was sutured by suture thread.About 10 days after the inoculation of the pancreatic cancer cell line,grouping was carried out by the body weight. Ten animals were assignedto each group, and were administered with a sham drug (non-treatmentgroup), comparative example 3, and test example 2, once every sevendays, three times in total. The dose was 20 mg/kg on the basis of SN-38.The animals were observed for a total of 28 days, and the tumor size andweight were measured by autopsy on day 28. The results are shown FIG. 5.

As a result of measurement of tumor inhibitory effect, the tumor weightof the non-treatment group (negative control group) was on average0.46±0.17g, the tumor weight of the monolayer micelle composition(comparative example 3) treatment group was 0.37±0.09g, and the tumorweight of the bilayer particle composition (test example 2) treatmentgroup was 0.21±0.05g. Therefore, the bilayer particle composition showeda tumor inhibitory effect of 55% or more compared with the monolayermicelle, and thus a very excellent tumor inhibitory effect.

Example 12 Pharmacokinetic Test in Beagle Dogs

The pharmacokinetic characteristics of the monolayer micelle composition(comparative example 3) were compared with those of the bilayer particlecomposition (test example 2) in beagle dogs. Male beagle dogs weighing7-10 kg were divided into two groups, three dogs per each group,according to the body weight, and were intravenously administered with amonolayer micelle composition (comparative example 2) and a bilayerparticle composition (test example 2) at 0.5 mg/kg on the basis of SN-38for 10 minutes infusion. Blood samples were taken at 0.33, 0.67, 1, 1.5,2, 4, 8, 12, 24, 36 hours after the end of the administration, and theplasma obtained by centrifuging the blood was pretreated by thefollowing method to measure the drug concentration in plasma. For samplepretreatment, 20 μL of S-(+)-camptothecin (500 ng/mL, dissolved inacetonitrile) as an internal standard substance was first added to 100μL of plasma, and 500 μL of acetonitrile was further added, followed byvortex-mixing for 30 seconds. After the mixture was centrifuged at12,000 rpm for 3 minutes, the supernatant was taken, and was injected 2μL into an LC-MS/MS system (API-5,000 model, AB Sciex). Separation wascarried out while the column was Gemini C18 (3 μm, 2.0×50 mm,Phenomenex, USA), the mobile phase was a 50% acetonitrile solutioncontaining 0.1% formic acid, and the flow rate was 0.25 mL/min. MS/MSdetection conditions were positive ion mode, and SN-38 glucuronide wasdetected at m/z 569.3→393.2, and the internal standard was detected atm/z 349.2→305.2.

The blood drug concentration over time after administration is shown inFIG. 6, and pharmacokinetic parameters therefor are shown in Table 9.

TABLE 9 Pharmacokinetic parameter comparison between monolayer micelleparticle composition and bilayer particle composition Dose Cmax TmaxAUCt Relative No Micelle type (mg/kg) (ng/mL) (hr) (ng · hr/mL) BA(%)Comparative Monolayer 0.5 25.30 ± 5.12 0.33 ± 0.00 131.84 ± 34.94 —example 3 micelles Test example 2 Double core- 0.5 66.25 ± 16.34 0.44 ±0.20 361.29 ± 96.25 275.6 shell *mean ± SD (n = 3)

In beagle dogs, SN-38 glucuronide produced directly from SN-38solubilized in the particles of the present invention was analyzed. Theresults confirmed that the bilayer particles of the present inventionshowed an increase in bioavailability by about 2.75 times, compared withthe monolayer micelles. It was determined from the above results thatthe bilayer particles of the present invention maximize the solubilityof the poorly soluble drug SN-38 in vivo.

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a preferred embodiment anddoes not limit the scope of the present invention.

What is claimed is:
 1. A particle comprising: i) a hydrophobiccamptothecin-based compound; ii) a hydrophilic camptothecin-basedcompound; and iii) an amphiphilic block copolymer composed of ahydrophobic block and a hydrophilic block.
 2. The particle of claim 1,wherein the hydrophobic camptothecin-based compound is at least oneselected from the group consisting of 7-ethyl-10-hydroxycamptothecin(SN-38), camptothecin, 10-hydroxycamptothecin, and a pharmaceuticalacceptable salt thereof.
 3. The particle of claim 1, wherein thehydrophilic camptothecin-based compound is at least one selected fromirinotecan, topotecan, belotecan, exatecan, lurtotecan, sinotecan,rubitecan, 9-nitrocamptothecin, 9-am inocamptothecin, gimatecan,BNP-1530, DB-67, BN-80915, BN-80927, a pharmaceutically acceptable saltthereof, a glucuronide metabolite thereof, and a glucuronide metaboliteof the hydrophobic camptothecin-based compound.
 4. The particle of claim1, wherein the amphiphilic block copolymer is composed of A-B or A-B-Ablocks, (a) wherein A is a hydrophilic polymer, which is monomethoxypolyethylene glycol, dimethoxy polyethylene glycol, polyethylene glycol,polypropylene glycol, monomethoxy polypropylene glycol, polyethyleneoxide, polyacrylic acid, or a polymer thereof; and (b) wherein B is ahydrophobic polymer, which is polylactic acid, polylactide, polyglycolicacid, polyglycolide, a polylactic acid-co-glycolic acid copolymer,polymandelic acid, polycaprolactone, polydioxan-2-one, polyglutamicacid, polyaspartic acid, polyornithine, polyorthoester, a derivativethereof, or a copolymer of two or more compounds selected therefrom. 5.The particle of claim 4, wherein the number average molecular weight ofthe hydrophilic polymer A is 500-10,000 Da.
 6. The particle of claim 4,wherein the number average molecular weight of the hydrophobic polymer Bis 500-10,000 Da.
 7. The particle of claim 1, wherein the weight ratioof the hydrophobic camptothecin-based compound and the hydrophiliccamptothecin-based compound is 1:10 to 10:1.
 8. The particle of claim 1,wherein the weight ratio of the sum of the hydrophobiccamptothecin-based compound and the hydrophilic camptothecin-basedcompound and the amphiphilic block copolymer is 1:200 to 10:1.
 9. Theparticle of claim 1, wherein the number average particle size of theparticle is 10-500 nm.
 10. The particle of claim 1, wherein the particlehas a double core-shell structure comprising the following: (a) an innercore-shell containing the hydrophilic camptothecin-based compound andthe hydrophobic camptothecin-based compound; and (b) an outer core-shellcontaining the amphiphilic block copolymer.
 11. The particle of claim 1,wherein the particle has a bilayer micelle structure.
 12. Apharmaceutical composition for treating cancer, comprising the particleof claim 1 and a pharmaceutically acceptable carrier.
 13. A method fortreating cancer, the method comprising administering the pharmaceuticalcomposition of claim 12 to a subject.
 14. A method for manufacturing aparticle, the method comprising: (a) forming an inner core-shellcontaining a hydrophobic camptothecin-based compound and a hydrophiliccamptothecin-based compound; and (b) forming an outer core-shellcontaining an amphiphilic copolymer.
 15. The method of claim 14, whereinstep (a) comprises mixing the hydrophobic camptothecin-based compoundand a hydrophilic camptothecin-based compound in an organic solvent; andwherein step (b) comprises mixing the inner core-shell and theamphiphilic block copolymer in an aqueous solvent.
 16. The method ofclaim 14, wherein step (a) comprises mixing a basic aqueous solution, inwhich a hydrophobic camptothecin compound is dissolved, and an aqueoussolution, in which a hydrophilic camptothecin compound is dissolved; andwherein step (b) comprises mixing the inner core-shell and theamphiphilic block copolymer in an aqueous solvent.
 17. The method ofclaim 14, wherein step (a) comprises mixing the hydrophobiccamptothecin-based compound and the hydrophilic camptothecin-basedcompound in an organic solvent; and wherein step (b) comprises mixing amixture of the hydrophobic camptothecin-based compound and thehydrophilic camptothecin-based compound with the amphiphilic blockcopolymer in an organic solvent.
 18. The method of claim 14, wherein theorganic solvent is a mixture solvent containing at least one selectedfrom a C1-05 alcohol, an alkyl acetate, acetone, acetonitrile,chloroform, benzene, toluene, xylene, acetone, a fluoroalkane, pentane,hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane,diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, diisopropylether, 2-chloropropane, 1-chloropropane, chlorobenzene, benzene, diethylether, diethyl sulfide, dichloromethane, 1,2-dichloroethane, aniline,diethyl amine, an ether, carbon tetrachloride, and tetrahydrofuran(THF).
 19. The method of claim 16, wherein the basic solution includesat least one selected from the group consisting of an inorganic alkali,an alkali salt of an organic acid, and an alkyl amine, the inorganicalkali comprising sodium hydroxide, potassium hydroxide, sodiumdihydrogenphosphate, potassium dihydrogenphosphate, magnesium hydroxide,sodium carbonate, and sodium hydrogencarbonate.